Table 2.

Assays to characterize nuclease off-target activity.

Assay Category	Assay Name	* Specialized laboratory technique(s) required?	Bioinformatics required?
Bulk Off-Target Activity	Viability Staining	No	No
	GFP Loss	No	No
	Cell Cycle Disruption	No	No
Phenotype Transformation	Soft Agar	No	No
	Clonal Barcoding	Yes	Yes
Off-Target Site Prediction	In silico prediction tools	No	Yes
	SELEX	Yes	Yes
	In vitro cleavage	Yes	Yes
	IDLV Trapping	Yes	Yes
	ChIP-Seq	Yes	Yes
Analysis of Clonal Populations	aCGH	Yes	Yes
	Whole Genome/Exome Sequencing	Yes	Yes

^*In defining laboratory techniques, we assume that researchers working with nucleases are familiar with basic cell transfection, detecting expression of fluorescent proteins, and cell culture protocols, but not more specialized procedures such as viral production or immunoprecipitation.